| Objectives:Glioma is the most common malignant tumor of the nervous system.The treatment effect is poor and the patient’s survival time is short.More in-depth research on the pathogenesis and treatment methods of gliomas are needed.Thyroid hormone is an important endocrine hormone,promotes organ development,regulates body metabolism,and is closely related to tumor occurrence and patient prognosis.Clinical glioma patients often complicated with hypothyroidism,which affects the quality of life and survival prognosis of patients.Hormone supplementation is required.Because Tetraiodothyronine(T4)is a tumor-promoting factor,supplementation of T4 has the risk of promoting tumor proliferation;and for the relationship between 3,5,3’-Triiodothyronin(T3)and gliomas,current research conclusions are inconsistent,it is not clear whether T3 can be used instead of T4 for hormone supplement therapy,and more researches are needed on the relationship between T3 and glioma.This study carried out experiments from the three levels:bioinformatics analysis,cell experiments and animal experiments to study the relationship between T3 and glioma cell proliferation and apoptosis and its mechanism,and to provide experimental basis for the selection of hormone supplement for glioma patients complicated with hypothyroidism in clinic.Methods:Bioinformatics Analysis1.The expressions of THRA and THRB in human normal tissues were studied by Gene Database of NCBI platform.2.The expressions of THRA,THRB and DIO2 in different types of tumors were studied by UALCAN platform.3.The effects of THRA and THRB expressions on the survival time of glioma patients were analyzed in TCGA database by GEPIA platform.4.Using the STRING database,the top 50 genes with the highest correlations with THRA and THRB were calculated through Protein-Protein Interaction network analysis,and the DAVID database was used to perform GO analysis and KEGG pathway enrichment analysis on the related genes.5.The genetic variations of THRA and THRB in human tumor tissues and cell lines were analyzed by the cBioPortal platform.The study on the relationship between the expression levels of THRA and THRB and the pathological grade of glioma1.Clinical adult glioma surgical specimens were collected(low grade 15 cases,high grade 15 cases).2.The expressions of THRA and THRB in gliomas were detected by RT-qPCR.The effect of T3 on proliferation and apoptosis of glioma cells(in vitro)1.The experiment was divided into blank control group and different concentrations(0-100 μM)NaOH(T3 solvent)experimental groups.Cell proliferation was detected by CCK-8.2.The experiment was divided into blank control group and T3 experimental groups of different concentrations(0-100 ng/ml),in the T3 experimental group,10μM NaOH was added as T3 solvent.Cell proliferation was detected by CCK-8 and apoptosis was detected by flow cytometry with Annexin V-FITC/PI double staining.The study on the role of THRA and THRB in T3 regulating proliferation and apoptosis of glioma Cells1.The experiment was divided into HS683 group,HS683+T3 group,A172 group and A172+T3 group,the concentration of T3 was 10 ng/mL.The cell cycle changes were detected by flow cytometry;Western blot was adopted to detect the expressions of THRA and THRB,proliferation pathway related proteins p-ERK and p-AKT,and cell cycle related factors Cyclin D1,Cyclin E,CDK2 and CDK4,apoptosis related proteins Caspase-3,Bcl-2 and Bax.2.RNA interference experiment I.The experiment was divided into HS683+si-NC+T3 group,HS683+si-THRA+T3 group,A172+T3 group and A172+si-THRA+T3 group.The concentration of T3 was 10 ng/mL,the cell proliferation was detected by CCK-8;the apoptosis was detected by Annexin V-FITC/PI double staining flow cytometry;the cell cycle changes were detected by flow cytometry;Western blot was adopted to detect the expression of THRA,proliferation pathway related proteins p-ERK and p-AKT,and cell cycle related factors Cyclin D1,Cyclin E,CDK2 and CDK4,apoptosis related proteins Caspase-3,Bcl-2 and Bax.3.RNA interference experiment Ⅱ.The experiment was divided into HS683+si-NC+T3 group,HS683+si-THRB+T3 group,A172+si-NC+T3 group and A172+si-THRB+T3 group.The concentration of T3 was 10 ng/mL,the cell proliferation was detected by CCK-8;the apoptosis was detected by Annexin V-FITC/PI double staining flow cytometry;the cell cycle changes were detected by flow cytometry;Western blot was adopted to detect the expression of THRB,proliferation pathway related proteins p-ERK and p-AKT,and cell cycle related factors Cyclin D1,Cyclin E,CDK2 and CDK4,apoptosis related proteins Caspase-3,Bcl-2 and Bax.The effect of T3 on proliferation and apoptosis of glioma cells(in vivo)The experiment was divided into control group(HS683/A172)and experimental group(T3 treatment).HS683 and A172 cells were subcutaneously tumorized in nude mice respectively,and the size and volume of tumors were measured weekly.After 2 weeks of T3 treatment,tumors were harvested,the change of cell proliferation ability was detected by EdU method,the cell apoptosis was detected by TUNEL method,the expressions of THRA and THRB in tumor tissues were detected by Western blot.Results:Bioinformatics Analysis1.The expressions of THRA in brain and ovary were higher than that in other tissues,and the expression of THRB in brain was higher than that in other tissues.2.Compared with normal tissues,THRA and THRB were generally down-regulated in different types of tumors,but up-regulated in some tumors,and the expressions varied greatly among different samples.3.High expression of THRA was positively correlated with the survival time of patients with low grade glioma;high expression of THRB was trended to positively correlated with the prolongation of survival time of patients with low grade glioma;while high expressions of THRA or THRB did not reduce the survival time of patients with glioblastoma multiforme.4.THRA and THRB were closely related to the occurrence and progression of tumors:THRA and its related genes were highly clustered in the "Thyroid hormone signaling pathway","Transcriptional dysregulation in cancer","Estrogen signaling pathway","Endocrine resistance" and "Non-small cell Lung Carcinoma".THRB and its related genes were highly clustered in the "Thyroid signaling pathway","Transcriptional dysregulation in cancer","Ubiquitin-mediated proteolysis","Non-small cell Lung Carcinoma",and "Thyroid cancer".5.There were different degrees of genetic variations and gene mutations of THRA and THRB in tumor samples and experimental cell lines:low grade glioma(THRA 0.7%;THRB 1.1%);glioblastoma multiformation(THRA 1.1%;THRB 0.8%);experimental cell lines(THRA 5%;THRB 1.1%);TCGA pan-cancer(THRA 3%;THRB 1.6%).The study on the relationship between the expression levels of THRA and THRB and the pathological grade of gliomaCompared with the low grade glioma group,the mean expressions of THRA and THRB in the high grade glioma group decreased,but there were no statistical differences between the two groups.The effects of T3 on proliferation and apoptosis of glioma cells(in vitro)1.NaOH inhibited the proliferation of glioma cells:compared with the blank control group,the cell survival rate of 100 μM NaOH group decreased,there was statistical difference between the two groups.2.T3 inhibited glioma cell proliferation and induced glioma cell apoptosis:compared with the solvent control group,the cell survival rate decreased in 1 ng/mL T3 group,10 ng/mL T3 group and 100 ng/mL T3 group,and there were statistical differences between the two groups.Compared with the solvent control group,the proportion of apoptosis in 10 ng/mL T3 experimental group increased,and there was statistical difference between the two groups.The study on the role of THRA and THRB in inhibiting proliferation and inducing apoptosis of glioma cells by T31.Compared with the blank control group(HS683/A172),the proportions of G1 phase in HS683 and A172 cells in were increased,the expressions of THRA and THRB were up-regulated,the expressions of p-ERK,p-AKT,Cyclin D1,Cyclin E,CDK2,CDK4 were down-regulated,and the expression of Caspase-3 was up-regulated in T3 experimental group.There were statistical differences between the two groups,but there were no significant change in the expressions of Bcl-2 and Bax.2.Compared with(HS683/A172)+si-NC+T3 group,the cell proliferation rate increased,the apoptosis proportion decreased,the proportion of G1 phase decreased,the expression of THRA down-regulated,the levels of p-ERK and p-AKT increased,the expression of Cyclin D1,Cyclin E,CDK2,CDK4 up-regulated,and the expression of apoptosis related protein Caspase-3 down-regulated in the(HS683/A172)+si-THRA+T3 group,there were significant differences between the two groups.but there were no significant changes in the expressions of Bcl-2 and Bax.3.Compared with(HS683/A172)+si-NC+T3 group,the cell proliferation rate increased,the apoptosis proportion decreased,the proportion of G1 phase decreased,the expression of THRB down-regulated,the levels of p-ERK and p-AKT increased,the expressions of Cyclin D1,Cyclin E,CDK2,CDK4 up-regulated,and the expression of apoptosis-related protein Caspase-3 down-regulated in the(HS683/A172)+si-THRB+T3 group,there were significant differences between the two groups.but there were no significant changes in the expressions of Bcl-2 and Bax.The effects of T3 on proliferation and apoptosis of glioma cells(in vivo)Compared with the blank control group(HS683/A172),the mean transplantation tumor volumes in the experimental group((HS683/A172)+T3)were reduced,the ability of cell proliferation decreased and cell apoptosis proportion increased,and the expressions of THRA and THRB were up-regulated.Conclusions:1.The overall expressions of THRA and THRB are down-regulated in gliomas,which are non-tumor promoting factors and are related to tumor occurrence and progression;there are varying degrees of THRA and THRB genetic variations in different types of tumors and experimental cell lines.2.Compared with low grade glioma group,the overall expressions of THRA and THRB in high grade glioma samples decrease,but there are no significant statistical differences between the two groups.3.NaOH can affect the proliferation of experimental cells,the results of T3 experiments need to consider the concentration of the solvent NaOH.4.T3 inhibits the proliferation of gliomas.One of the mechanism is that T3 induces the up-regulation of THRA and THRB expression,thereby inhibiting the expressions of p-ERK and p-AKT,inhibiting the expressions of Cyclin D1,Cyclin E,CDK2,and CDK4,and inducing cells to appear G1 phase arrest,inhibits cell division.5.T3 induces apoptosis in gliomas.One of the mechanism is that T3 induces the up-regulation of THRA and THRB expressions,activates the apoptosis pathway,and promotes the expression of downstream apoptosis factor Caspase-3 through non-mitochondrial apoptosis pathways,and then induces cell apoptosis.6.T3 supplementation may be considered clinically in gliomas patients with hypothyroidism,The detection of genetic variations and expressions of THRA and THRB are helpful to evaluate the effect of T3 supplementation. |
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